MECHANISMS THAT LIMIT THE DIVERSITY OF ANTIBODY - 3 SEQUENTIALLY ACTING MECHANISMS THAT FAVOR THE SPONTANEOUS PRODUCTION OF GERMLINE ENCODED ANTI-PHOSPHATIDYL CHOLINE

Antibody to phosphatidyl choline (PtC) is produced spontaneously in mi ce, by approximately 2-10% of naturally occurring CD5+ (B1) B cells in the peritoneum. Much of this antibody is encoded by the V(H)11 gene a ssociated with a specific V(kappa)9 gene. Constraints on the size and structure of the H chain CDR3 have been defined from nucleotide sequen ces of genes expressed by hybridomas and lymphomas derived from adult mice. All employ J(H)1 and all encode tyrosine as the first amino acid in CDR3, which is either nine or 10 amino acids long; the last six ar e always the same, start with tyrosine, and are rich in aromatic amino acids. Those with nine amino acids in CDR3 have glycine or serine in the second position and asparagine, serine or proline in the third; th ose with 10 have an additional aspartate or glycine inserted after the first tyrosine. DSP2 genes are used by 80% and DFL16 by 20%. Producti vely rearranged V(H)11 genes in neonates and in 18 day fetal liver dis play a greater, but still limited degree of diversity. All four J(H) g enes are used and the length of CDR3 varies from three to 12 amino aci ds, but the first is tyrosine in 58 of 61 and DSP2 genes are used by 8 0% of these productive VDJ assemblies. Non-productive rearrangements O f V(H)11 in fetal liver show a different pattern; 41% use DFL16 genes and only 40% have a TAx codon in the first position of CDR3. All rearr angements show evidence of a bias in favor of joining the V(H)11 gene to D genes at positions of matching nucleotide overlap. The data are i nterpreted as indicating the existence of three sequentially acting me chanisms that lead to the spontaneous appearance of anti-PtC antibodie s; a biased rearrangement of germ line elements, selective survival of pre-B cells, and classical, antigen-driven, clonal selection.